This invention relates to fragments of the tumor associated gene product MAGE-3 which bind to and are presented to T lymphocytes by HLA class II molecules. The peptides, nucleic acid molecules which code for such peptides, as well as related antibodies and CD4+ T lymphocytes, are useful, inter alia, in diagnostic and therapeutic contexts.
The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is complex. An important facet of the system is the T cell response, which in part comprises mature T lymphocytes which are positive for either CD4 or CD8 cell surface proteins. T cells can recognize and interact with other cells via cell surface complexes on the other cells of peptides and molecules referred to as human leukocyte antigens (xe2x80x9cHLAsxe2x80x9d) or major histocompatibility complexes (xe2x80x9cMHCsxe2x80x9d). The peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecule. See Male et al., Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters 6-10. The interaction of T cells and complexes of HLA/peptide is restricted, requiring a specific T cell for a specific complex of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. The mechanisms described above are involved in the immune system""s response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities.
The T cell response to foreign antigens includes both cytolytic T lymphocytes and helper T lymphocytes. CD8+ cytotoxic or cytolytic T cells (CTLs) are T cells which, when activated, lyse cells that present the appropriate antigen presented by HLA class I molecules. CD4+ T helper cells are T cells which secrete cytokines to stimulate macrophages and antigen-producing B cells which present the appropriate antigen by HLA class II molecules on their surface.
The mechanism by which T cells recognize alien materials also has been implicated in cancer. A number of cytolytic T lymphocyte (CTL) clones directed against autologous melanoma have been described. In some instances, the antigens recognized by these clones have been characterized. In De Plaen et al., Immunogenetics 40:360-369 (1994), the xe2x80x9cMAGExe2x80x9d family, a family of genes encoding tumor specific antigens, is described. (See also PCT application PCT/US92/04354, published on Nov. 26, 1992.) The expression products of these genes are processed into peptides which, in turn, are expressed on cell surfaces. This can lead to lysis of the tumor cells by specific CTLs. The genes are said to code for xe2x80x9ctumor rejection antigen precursorsxe2x80x9d or xe2x80x9cTRAPxe2x80x9d molecules, and the peptides derived therefrom are referred to as xe2x80x9ctumor rejection antigensxe2x80x9d or xe2x80x9cTRAsxe2x80x9d. See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254: 1643 (1991), for further information on this family of genes. Also, see U.S. Pat. No. 5,342,774.
In U.S. Pat. No. 5,405,940, MAGE nonapeptides are taught which are presented by the HLA-A1 molecule. Given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.
In U.S. Pat. No. 5,591,430, additional isolated MAGE-3 peptides are taught which are presented by the HLA-A2 molecule. Therefore, a given TRAP can yield a plurality of TRAs.
The foregoing references describe isolation and/or characterization of tumor rejection antigens which are presented by HLA class I molecules. These TRAs can induce activation and proliferation of CD8+ cytotoxic T lymphocytes (CTLs) which recognize tumor cells that express the tumor associated genes (e.g. MAGE genes) which encode the TRAs.
The importance of CD4+ T lymphocytes (helper T cells) in antitumor immunity has been demonstrated in animal models in which these cells not only serve cooperative and effector functions, but are also critical in maintaining immune memory (reviewed by Topalian, Curr. Opin. Immunol. 6:741-745, 1994). Moreover, several studies support the contention that poor tumor-specific immunity is due to inadequate activation of T helper cells.
It has recently been demonstrated that the tyrosinase gene encodes peptides which are presented by HLA class II molecules to stimulate CD4+ T lymphocytes (Topalian et al., 1994; Yee et al., J. Immunol. 157:4079-4086, 1996; Topalian et al., J. Exp. Med. 183:1965-1971, 1996).
It now has been discovered that the MAGE-3 gene encodes additional tumor rejection antigens which are HLA class II binding peptides. These peptides, when presented by an antigen presenting cell having an HLA class II molecule, effectively induce the activation and proliferation of CD4+ T lymphocytes.
The invention is elaborated upon in the disclosure which follows.
The invention provides isolated MAGE-3 peptides, and functional variants thereof, which bind HLA class II molecules. The invention also provides isolated nucleic acid molecules encoding such peptides, expression vectors containing those nucleic acid molecules, host cells transfected with those nucleic acid molecules, and antibodies to those peptides and complexes of the peptides and HLA class II antigen presenting molecules. T lymphocytes which recognize complexes of the peptides and HLA class II antigen presenting molecules are also provided. Kits containing the foregoing molecules additionally are provided. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of MAGE-3.
According to one aspect of the invention, an isolated HLA class II-binding peptide is provided. The isolated peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. In certain embodiments, the isolated HLA class II-binding peptide comprises the amino acid sequence of SEQ ID NO:9. In preferred embodiments, the isolated HLA class II-binding peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. In particularly preferred embodiments, the isolated peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. More preferably, the isolated peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4. In other embodiments of the invention, the isolated HLA class II-binding peptide is non-hydrolyzable. Preferred non-hydrolyzable peptides are selected from the group consisting of peptides comprising D-amino acids, peptides comprising a -psi[CH2NH]-reduced amide peptide bond, peptides comprising a -psi[COCH2]-ketomethylene peptide bond, peptides comprising a-psi[CH(CN)NH]-(cyanomethylene)amino peptide bond, peptides comprising a-psi[CH2CH(OH)]-hydroxyethylene peptide bond, peptides comprising a -psi[CH2O]-peptide bond, and peptides comprising a -psi[CH2S]-thiomethylene peptide bond.
According to another aspect of the invention, a composition comprising an isolated MAGE-3 HLA class I-binding peptide and an isolated MAGE-3 HLA class II-binding peptide is provided. In certain embodiments the isolated MAGE-3 HLA class II-binding peptide in the composition comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. Preferably, the isolated MAGE-3 HLA class II-binding peptide in the composition consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. More preferably, the isolated MAGE-3 HLA class II-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4.
According to another aspect of the invention, an isolated nucleic acid encoding any of the foregoing HLA class II-binding peptide peptides is provided. Preferably the nucleic acid comprises SEQ ID NO:13.
According to still another aspect of the invention, expression vectors are provided. The expression vectors comprise any of the foregoing isolated nucleic acids operably linked to a promoter. In preferred embodiments, the nucleic acid comprises SEQ ID NO:13. In other embodiments, the expression vector further comprise a nucleic acid which encodes an HLA-DRB1/13 molecule.
According to yet another aspect of the invention, host cells transfected or transformed with any of the foregoing expression vectors are provided. Host cells which express an HLA-DRB1/13 molecule, and which are transfected or transformed with any of the foregoing expression vectors are also provided.
According to another aspect of the invention, methods for enriching selectively a population of T lymphocytes with CD4+ T lymphocytes specific for a MAGE-3 HLA class II-binding peptide are provided. The methods include contacting an isolated population of T lymphocytes with an agent presenting a complex of the MAGE-3 HLA class II-binding peptide and an HLA class II molecule in an amount sufficient to selectively enrich the isolated population of T lymphocytes with the CD4+ T lymphocytes. In certain embodiments, the agent is an antigen presenting cell contacted with a MAGE-3 protein or an HLA class II binding fragment thereof. In preferred embodiments, the HLA class II molecule is an HLA-DRB1/13 molecule and the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
According to a further aspect of the invention, methods for diagnosing a disorder characterized by expression of MAGE-3 are provided. The methods include contacting a biological sample isolated from a subject with an agent that is specific for the MAGE-3 HLA class II binding peptide, and determining the interaction between the agent and the MAGE-3 HLA class II binding peptide as a determination of the disorder. In certain embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. In preferred embodiments, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
According to another aspect of the invention, methods for diagnosing a disorder characterized by expression of a MAGE-3 HLA class II-binding peptide which forms a complex with an HLA class II molecule are provided. The methods include contacting a biological sample isolated from a subject with an agent that binds the complex; and determining binding between the complex and the agent as a determination of the disorder. In some embodiments the HLA class II molecule is an HLA-DRB1/13 molecule, such as HLA-DRB1/1301 or HLA-DRB1/1302, and the MAGE-3 HLA class II-binding peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. Preferably the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
Methods for treating a subject having a disorder characterized by expression of MAGE-3 are provided in another aspect of the invention. The methods include administering to the subject an amount of a MAGE-3 HLA class II-binding peptide sufficient to ameliorate the disorder. In certain embodiments the MAGE-3 HLA class II-binding peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. Preferably the peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
According to still another aspect of the invention, methods for treating a subject having a disorder characterized by expression of MAGE-3 are provided. The methods include administering to the subject an amount of a MAGE-3 HLA class I-binding peptide and an amount of a MAGE-3 HLA class II-binding peptide sufficient to ameliorate the disorder. In certain embodiments, the MAGE-3 HLA class II-binding peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. Preferably the peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
According to yet another aspect of the invention, methods for treating a subject having a disorder characterized by expression of MAGE-3 are provided. The methods include administering to the subject an amount of an agent which enriches selectively in the subject the presence of complexes of an HLA class II molecule and a MAGE-3 HLA class II-binding peptide, sufficient to ameliorate the disorder. Preferably the HLA class II molecule is an HLA-DRB1/13 molecule, such as HLA-DRB1/1301 or HLA-DRB1/1302. In certain embodiments, the MAGE-3 HLA class II-binding peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. Preferably the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
Additional methods for treating a subject having a disorder characterized by expression of MAGE-3 are provided in another aspect of the invention. The methods include administering to the subject an amount of autologous CD4+ T lymphocytes sufficient to ameliorate the disorder, wherein the CD4+ T lymphocytes are specific for complexes of an HLA class II molecule and a MAGE-3 HLA class II-binding peptide. Preferably the HLA class II molecule is an HLA-DRB1/13 molecule, such as HLA-DRB1/1301 or HLA-DRB1/1302. In certain embodiments, the MAGE-3 HLA class II-binding peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. Preferably the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
According to another aspect of the invention, an isolated polypeptide is provided. The isolated polypeptide binds selectively a MAGE-3 HLA class II-binding peptide, provided that the isolated polypeptide is not an HLA class II molecule. In certain embodiments, the isolated polypeptide is an antibody and preferably is a monoclonal antibody. In other embodiments, the isolated polypeptide is an antibody fragment selected from the group consisting of a Fab fragment, a F(ab)2 fragment or a fragment including a CDR3 region selective for a MAGE-3 HLA class II-binding peptide.
According to still another aspect of the invention, an isolated CD4+ T lymphocyte is provided. The isolated CD4+ T lymphocyte selectively binds a complex of an HLA class II molecule and a MAGE-3 HLA class II-binding peptide. Preferably the HLA class II molecule is an HLA-DRB1/13 molecule. In some embodiments the MAGE-3 HLA class II-binding peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. Preferably the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
According to still another aspect of the invention, an isolated antigen presenting cell is provided. The isolated antigen presenting cell comprises a complex of an HLA class II molecule and a MAGE-3 HLA class II-binding peptide. Preferably the HLA class II molecule is an HLA-DRB1/13 molecule. In certain embodiments the MAGE-3 HLA class II-binding peptide comprises the amino acid sequence of SEQ ID NO:11, or a functional variant thereof. In preferred embodiments the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3, a peptide consisting of the amino acid sequence of SEQ ID NO:4, peptide consisting of the amino acid sequence of SEQ ID NO:9, peptide consisting of the amino acid sequence of SEQ ID NO:10, and a peptide consisting of the amino acid sequence of SEQ ID NO:11. More preferably, the MAGE-3 HLA class II-binding peptide is selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO:3 and a peptide consisting of the amino acid sequence of SEQ ID NO:4.
Methods for identifying functional variants of a MAGE-3 HLA class II binding peptide are provided according to another aspect of the invention. According to the methods, a MAGE-3 HLA class II binding peptide, an HLA class II binding molecule which binds the MAGE-3 HLA class II binding peptide, and a T cell which is stimulated by the MAGE-3 HLA class II binding peptide presented by the HLA class II binding molecule are selected. A first amino acid residue of the MAGE-3 HLA class II binding peptide is mutated to prepare a variant peptide. The binding of the variant peptide to HLA class II binding molecule and stimulation of the T cell are then determined, wherein binding of the variant peptide to the HLA class II binding molecule and stimulation of the T cell by the variant peptide presented by the HLA class II binding molecule indicates that the variant peptide is a functional variant. In preferred embodiments, the MAGE-3 HLA class II binding peptide comprises the amino acid sequence of SEQ ID NO:11. More preferably, the peptide consists of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In certain embodiments, the methods further include the step of comparing the stimulation of the T cell by the MAGE-3 HLA class II binding peptide and the stimulation of the T cell by the functional variant as a determination of the effectiveness of the stimulation of the T cell by the functional variant.
The invention also provides pharmaceutical preparations containing any one or more of the medicaments described above or throughout the specification. Such pharmaceutical preparations can include pharmaceutically acceptable diluent carriers or excipients.
These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.